Defects in ubiquitin (Ub) pathways are often responsible for cancer and devastating neurodegenerative diseases. Because of its central role in biological circuits and the potential for therapeutic intervention, the Ub system is an intense research area. Yet, Ub biology is highly complex?in humans it is supported by over 500 Ub ligases and 95 deubiquitinases. Currently, this complexity and our limited understanding of players and their interactions are a severe hindrance for targeted intervention in many diseases. Considering all possible ways to address the complexity, sample multiplexing in mass spectrometry-based proteomics arguably has the greatest potential to fundamentally revolutionize the throughput of these measurements. Through efforts within the previous grant cycle, the level of multiplexing was increased, facilitating the quantitative comparison of expression and conjugate levels for 10 samples. In this proposal, enabling advances in sample multiplexing will be explored and applied to study ubiquitin-dependent biology. In Aim 1, we will evaluate isobaric reagents that have multiple reporter ions such that a 16- and a 32-plex reagent set can be synthesized using relatively few heavy atoms (4 and 6, respectively). In Aim 2, two large obstacles to sample multiplexing will be addressed?sensitivity and proteome depth. Using a real-time database search algorithm, reporter ion quantification scans (SPS-MS3 scans) will only be collected when the MS2 scan is successfully matched to a peptide or PTM-containing peptide. This will be a very disruptive technology as MS2 scans are recorded at a blistering rate of 22 Hz. Highly optimized MS3 scans will then be collected based on any filter desired since the identity and modification state of all peptides will be known in real time. We expect dramatic improvements in the quality and depth of discovery proteomics experiments while reducing the analysis time by at least 3 fold. In Aim 3, we will explore two new multiplexing workflows based on the technologies developed in the first two aims. One workflow will be dedicated to targeted proteomics where up to 32-plex experiments targeting hundreds of peptides will be developed. In these assays, triggering peptides and a real-time database search will obviate the need for retention time scheduling or precursor detection in survey scans prior to MS3 analysis using pre-selected SPS ions. Using panels of trigger peptides, assays to monitor the protein levels in six ubiquitin-dependent pathways will be created. The second workflow will be dedicated to discovery proteomics where up to 32-plex experiments will be conducted for full proteome or conjugate analysis in as little as 24 hr. This will be applied to characterize CRISPR deletions in the mTOR amino acid sensing pathway and specifically the mechanism of action of the GATOR2 complex where 3 ubiquitin ligases of unknown function are present. The realization of all three aims ushers in a paradigm shift in Quantitative Proteomics where hundreds to thousands of samples can be accurately compared across entire proteomes or in targeted assays in a matter of days or weeks using up to 32-plex reagent sets and real-time database searching.